JPH0581569B2 - - Google Patents

Abstract

Healing an external wound or regenerating bone of a mammal by administering to the mammal a composition containing purified platelet-derived growth factor and purified insulin-like growth factor.

Description

Claim 1: A composition for wound healing and bone regeneration comprising platelet-derived growth factor (PDGF) and insulin-like growth factor I (IGF-1) in a weight-to-weight ratio of 1:4 to 25:1.

2. A composition according to claim 1, wherein the ratio is from 1:2 to 10:1.

3. A composition according to claim 1, wherein the ratio is from 1:1 to 2:1.

4. Production of a composition for wound healing and bone regeneration comprising mixing platelet-derived growth factor (PDGF) and insulin-like growth factor I (IGF-1) in a weight-to-weight ratio of 1:4 to 25:1. Method.

BACKGROUND OF THE INVENTION The present invention is based on Antoniades et al., U.S. Patent Application No.
No. 930762: Title of invention “Healing of trauma
External Wounds): Continuation-in-part application filed November 14, 1986, the contents of which are also incorporated herein by reference.

The present invention relates to wound healing.

Growth factors are polypeptide hormones that stimulate specific target cell populations. Examples of growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), tranforming growth factor (TGF-β), epidermal growth factor (EGF), and fibroblastic growth factor. (FGF).
PDGF is a cationic, thermostable protein found in the granules of platelets in the bloodstream and is known to stimulate in vitro protein synthesis and collagen production in fibroblasts. This factor is
and fibroblasts, smooth muscle cells and glial cells.
It is also known to act as a cell division and chemotactic factor in vitro.

The use of PDGF for in vivo wound healing has already been proposed. For example, Grotendorst (1984)
J. Trauma 24; 549-52 describes a Hunt-like structure impregnated with collagen gel and implanted on the back of a rat.
It has been described that the addition of PDGF to Schilling wire mesh chambers increased the amount of newly synthesized collagen. However, Leitzel
(1985) J. Dermatol. Surg. Oncol. 11:617−22
According to PDGF alone or FGF and EGF
When used in combination with hamsters, it was not possible to promote normal wound healing in hamsters.

Michaeli et al. (1984) “Soft and Hard Tissue Repair”
(Hunt, TK et al., eds., Praeger Publishers, New York, pp. 380-394) reported that a partially purified PDGF preparation obtained from platelet lystiplasma stimulated angiogenesis when implanted into the cornea of domestic rabbits. are doing. Since PDGF is not an angiogenic growth factor, the above authors reported that their partial purification
It is suggested that an unknown factor in the PDGF preparation was involved in this angiogenic effect.

SUMMARY OF THE INVENTION In general, in one aspect, the present invention relates to techniques for healing trauma in a mammal, such as a human patient, by applying a composition comprising an effective amount of purified PDGF and purified IGF-I to the wound. The composition contributes to wound healing, at least in part, by stimulating epithelial and connective tissue proliferation and total protein and collagen synthesis.
Wound healing with the compositions of the present invention occurs in the absence of such treatment (i.e., without the use of external agents) or with pure PDGF alone or pure IGF-I alone. It is more effective compared to

In other embodiments, the invention applies purified PDGF and purified PDGF to an area of injured or broken bone in a patient.
The present invention relates to techniques for regenerating bone in mammalian, e.g. human, patients, applied by applying an effective amount of a composition of the invention comprising IGF-I. The composition contributes to wound healing, at least in part, by stimulating connective tissue, bone and cementum proliferation and total protein and collagen synthesis. Bone regeneration using the compositions of the invention occurs in the absence of such treatment (i.e., without external agents) or with pure PDGF alone or pure IGF-I alone. It is more effective compared to

In both of the above embodiments, in a preferred embodiment of the invention, the composition is provided in a pharmaceutically acceptable carrier material, such as a commercially available inert gel or solution (e.g., saline supplemented with albumin or methylcellulose). Purified PDGF and purified IGF-I
(both are commercially available). Most preferably purified
PDGF and purified IGF-I have a weight-to-weight ratio of
1:4 to 25:1, preferably 1:2 to
10:1 and more preferably 1:1 or 2:
1 is mixed. Purified PDGF and purified IGF-I
may be obtained from human platelets or by recombinant DNA technology. Therefore, PDGF and IGF-I
The term refers both to platelets of a mammal, preferably a primate, or derived from genetically modified material, most preferably the primate is a human, but may also be a chimpanzee or other primate. Recombinant PDGF may be a heterodimer obtained by inserting DNA sequences encoding both subunits into cultured prokaryotic or eukaryotic cells and then treating the cells with the translated subunits, or alternatively, only one of the subunits (preferably DNA encoding beta or “2” chain) is inserted into cells, which are then cultured to produce homodimeric PDGF (
PDGF-1 or PDGF-2 homodimer) may also be produced.

In the present invention, the term purified means that 95% or more by weight of PDGF or IGF is present before mixing with other ingredients.
-I, meaning that other naturally occurring proteins, lipids and carbohydrates are substantially absent.

Purified protein preparations generally give only one major band for each subunit of PDGF or IGF-I on polyacrylamide gels. Most preferably, the purified product used in the composition of the invention
PDGF or IGF-I is pure as determined by amino-terminal amino acid sequence analysis.

The compositions of the present invention provide a rapid and effective method for healing trauma in mammals, such as bedsores, lacerations, and burns. This composition can be used for natural healing (i.e. when no external drugs are applied) or for pure
Enhances connective tissue formation compared to PDGF or IGF-I alone. Unlike pure PDGF alone, this composition increases new connective tissue by approximately 250% and increases epithelial tissue proliferation by approximately 95%. The resulting epithelial layer is not only thicker than in natural healing, but also has epithelial projections to connect it with new connective tissue.
It contains more projections, therefore it binds more tightly and has a greater effect on wound protection. Furthermore, there is less risk of scratches.

The compositions of the present invention provide a rapid and effective method for regenerating connective tissue and bone in mammals, such as humans with a history of periodontal ligament disease. This composition enhances connective tissue and bone formation compared to natural healing (ie, when no external drug is applied) or pure PDGF or IGF-I alone.

Other aspects and advantages of the invention will become apparent from the following description of the preferred embodiments and from the claims.

Description of preferred embodiments A preferred embodiment will be explained.

According to the present invention, PDGF/IGF-I mixtures prepared with a combination of purified PDGF and IGF-I are used to treat trauma, such as bedsores and burns, and to regenerate bone and connective tissue. IGF-I is
It is commercially available from Amgen (Thousand Oaks, Calif.) and Kabi (Sweden). Purified recombinant PDGF and purified PDGF derived from human platelets were purchased from PDGF, Inc. (Boston, USA).
Collaborative Research (Waltham, Mass.) and Amgen (Thousand Oaks, Calif.). Purified PDGF can also be prepared as follows: 500 to 1000 units of washed human platelet pellets are suspended in 1M NaCl (2 ml per platelet unit) and heated at 100°C for 15 minutes. The supernatant is separated by centrifugation and the precipitate is extracted with 1M NaCl.

The extracts were combined and dialyzed against 0.08M NaCl-0.01M sodium phosphate buffer (PH7.4).
Mix overnight at 4°C with CM-Sephadex C-50 equilibrated with this buffer. This mixture was then injected into a column (5 x 100 cm) and 0.08M
NaCl − 0.01M sodium phosphate buffer (PH
7.4), elute with 1M NaCl, and
Collect in 10 ml fractions.

Pool the active fractions and add 0.3M NaCl−
Dialyzed against 0.01M sodium phosphate buffer (PH7.4), centrifuged, 0.3M NaCl−0.01M
Pass through 2.5 x 25 cm of Blue Sepharose (Pharmacia) equilibrated with sodium phosphate buffer (PH 7.4) at 4°C. This column was washed with the same buffer and partially purified
Elute with a 1:1 solution of PDGF1M NaCl and ethylene glycol.

This partially purified PDGF fraction was purified using 1M NaCl.
diluted 1:1 with 1M acetic acid, dialyzed against 1M acetic acid, and lyophilized. Lyophilized samples were 0.8M NaCl−
1.2×40 dissolved in and equilibrated in 0.01M sodium phosphate buffer (PH7.4)
Pass through a CM-Sephadex C-50 column at cm. PDGF was then added to a NaCl gradient (from 0.08 to
Elute at 1M).

The active fractions are combined, dialyzed against 1M acetic acid, lyophilized and dissolved in a small amount of 1M acetic acid. 1.2 of which 0.5 ml was equilibrated with 1M acetic acid.
Place on a column of Biogel P-150 (100 to 200 mesh) x 100 cm. Then 1M PDGF
Elute with acetic acid and collect in 2 ml fractions.

Each active fraction containing 100 to 200 mg of protein is lyophilized, dissolved in 100 ml of 0.4% trifluoroacetic acid, and subjected to reversed-phase high performance liquid chromatography on a Phenyl Bonda Pack column (Waters). Linear gradient of acetonitrile (0
to 60%) to obtain purified PDGF.

PDGF with recombinant DNA technology is prepared as follows: Platelet-derived growth factor (PDGF) obtained from human platelets has two polypeptide sequences (PDGF-
1 and PDGF-2 polypeptides) (Antoniades, HN and Hunkapiller, M.
(1983) Science, 220:963-965). PDGF-1 is encoded by a gene located in chromosome 7 (Betsholtz, C. et al., Nature, 320:695-
699), and PDGF-2 is chromosomal 22 (Dalla
-Favera, R. (1982) Science, 218:686-688)
sis oncogene (Doolittle, R. et al. (1983) Science 221:275-277). The sis gene is derived from the simian sarcoma virus (Simian
It encodes the transformation protein of Sarcoma Virus (hereinafter referred to as SSV), and this protein is PDGF-
2 polypeptides. Human cell c- sis also encodes the PDGF-2 chain (Rao,
CD et al. (1986) Proc. Natl. Acad. Sci. USA, 83:
2392−2396). These two polypeptide chains of PDGF are encoded by different genes located in different chromosomes, so human PDGF is
Maya may consist of a disulfide-bonded heterodimer of PDGF-1 and PDGF-2, or two types of homodimers (a homodimer of PDGF-1 and a homodimer of PDGF-2).
It may consist of a mixture of two types of homodimers) or a mixture of the above heterodimer and two types of homodimers.

Mammalian cells in culture infected with SSV containing the gene encoding the PDGF-2 chain are known to synthesize PDGF-2 polypeptide and process it into disulfide-linked homodimers (Robbins, K. (1983) Nature, 305:605−
608). Furthermore, PDGF-2 homodimer is
Reacts with antiserum prepared against PDGF. Furthermore, the functional properties of the secreted PDGF-homodimer include stimulating DNA synthesis in fibroblasts in culture, inducing phosphorylation at tyrosine residues of a 185 kd cell membrane protein, and specific cell surface It is similar to that of platelet-derived PDGF in that it has the ability to compete with human ( ¹²⁵ I)-PDGF for binding to PDGF receptors (Owen,
A. et al. (1984) Science, 225:54-56). Similar properties have been shown for sis /PDGF-2 gene products from cultured normal human cells (e.g. human arterial endothelial cells) or for products from sis /PDGF-2 expressing human malignant cells (Antoniades , H. et al. (1985), Cancer Cells,
3:145-151).

Recombinant PDGF-2 homodimer is c- sis /
A clone of the PDGF-2 gene can be obtained by introducing it into mouse cells using an expression vector. The c- sis /PDGF-2 clone used for this expression was
Obtained from cultured normal human epithelial cells (Collins,
T. et al. (1985), Nature, 216:748-750).

Wound Healing To promote wound healing, PDGF/IGF-
The following experiment was conducted to examine the effectiveness of the I mixture.

Juvenile white Yorkshire pigs (Parson's Farm, Hadley, Mass.) weighing 10 to 15 kg were fasted for at least 6 hours before surgery and then anesthetized. The back and neck areas were cut, shaved, and washed with mild soap and water under sleeping conditions. The area to be traumatized was then disinfected with 70% alcohol.

A modified Castroviejo electrokeratome (manufactured by Storz, St. Louis, Missouri, modified by Brownells, Inc.) was used to create a wound measuring 1 cm x 2 cm with a depth of 0.5 mm. This wound resulted in complete removal of the epithelium and partial removal of the underlying dermis (corresponding to a second degree burn). Each wound has at least
Separated by 15 mm of healthy skin. Wounds receiving the same treatment were grouped together and separated by at least 3 cm from other groups. Wounds not treated with growth factors were at least
10cm apart.

Wounds were treated directly with a single application of the following growth factors dispersed in a biologically acceptable gel: 1) 500 ng pure human PDGF (purified by high performance liquid chromatography) or recombinant
PDGF only: 2) Each in combination with the following:
500ng pure PDGF: (a) 500ng EGF; (b) 500ng
EGF + 500 ng IGF-I; (c) 500 ng IGF-I.

Biopsy specimens were taken from day 3 to day 10 following wounding. Biopsy specimens for histological evaluation were taken in a wedge shape approximately 3 mm deep and preserved in 10% formalin. Specimens for biochemical analysis and autoradiography were collected using an electrokeratome. The final dimensions of the specimen are
It was 1.5mm x 10mm x 1.5mm. Three specimens were taken per wound for biochemical analysis and two specimens per wound for autoradiography. After specimens were collected, they were stored in chilled Eagle's modified basal medium (EMEM) supplemented with 10% calf serum. Biopsy specimens were analyzed as follows.

Autoradiography Biopsy specimens containing 15 μCi/ml ^3H -thymidine
0.3ml Eagle Modified Basic Medium (EMEM) +10%
After incubating with fetal bovine serum for 1 hour, the specimens were then washed twice with 10% formalin. Four micron sections were cut from the specimens using standard paraffin impregnation and embedding techniques. This was then deparaffinized, immersed in Nuclear Tract emulsion NTB-2 (Kodatsu), and exposed for two weeks. This was subsequently developed and stained with hematoxylin and eosin.

Autoradiograms of the stained specimens were recorded and scored by counting the total number of labeled (5 or more grains per cell) cells at evenly distributed points.

Histological Evaluation Specimens for histological examination were prepared using standard paraffin impregnation and embedding techniques. Four micron sections were made and stained with prefiltered Harris hematoxylin and alcoholic eosin before microscopic observation. For all specimens, two observers independently scored the sections at evenly distributed points.
The widths of the epithelial and connective tissue layers were measured using a grid placed in the eyepiece of the microscope, and the results were compared with the micrometer results observed under the same conditions and converted to mm.

In vitro metabolic labeling Biopsy specimens were treated with 0.3 ml of EMEM + 10% fetal bovine serum, 15 μCi/ml of ³ H-thymidine and 5 μCi/ml of
Transfer to separate tubes containing ^14C -leucine and incubate at 37°C.
It was incubated for 1 hour. At the end of the incubation period, the medium was removed and 0.5 ml of cold 5% perchloric acid was added to stop tissue metabolism. Next, this specimen was crushed into small pieces and washed three times, and the resulting precipitate was
Resuspend in 0.5 ml of 14.8 M ammonium hydroxide;
Crushed using ultrasound. After sonication, specimens were incubated in sealed test tubes at 45° C. for 16 to 24 hours, sonicated again, and radioactivity was measured using standard liquid scintillation methods with cross-channel counting.

DNA and protein measurements DNA measurements are described in Labarca et al. (1980) Anal.
Biochem. 120:344-52 with a modification. 50 μl of the tissue extract in concentrated ammonium hydroxide was mixed with 400 μl of a buffer solution containing 1 M sodium phosphate and 2 M sodium chloride (PH
7.0) and the pH of the resulting solution was adjusted to 7.4 with HCl. Thereafter, the final solution volume was made to 500 μl while keeping the pH at 7.4. This solution was then added to 2.5 ml of a buffer solution (0.05 M sodium phosphate, 2 M sodium chloride, PH=7.4) containing Hoechst dye (1.14 mg/ml). Fluorescence was induced at an excitation wavelength of 352 nm, and the intensity of emission was measured at 454 nm. Calf thymus DNA subjected to similar treatments was used to obtain the standard curve.

The protein content of tissue extracts in concentrated ammonium hydroxide was determined using the Bradford method.
(1976) Anal.Biochem.72:248-54) using bovine serum albumin as a standard.

Results Results from histological evaluation showed that wounds treated with purified human PDGF or recombinant PDGF in combination with IGF-I were significantly lower than untreated wounds, pure IGF
-I, the connective tissue and epithelial layers are thicker and more epithelial projections line these layers compared to wounds treated with pure PDGF or with PDGF in combination with other growth factors. It was shown that they are connected. No signs of scar development were observed until 6 weeks after treatment. Autoradiography showed an increased proportion of ³ H-thymidine-labeled cells in wounds treated with PDGF/IGF-I than in wounds treated with no treatment, PDGF alone, or IGF-I alone. Metabolic labeling results showed that ^3H -thymidine and ^14C -leucine uptake was greater in wounds treated with purified PDGF/IGF-I preparations than in untreated wounds, as well as in PDGF/IGF-I preparations. I treated wounds are DNA
It also had a high protein content.

Recombinant purified PDGF-2 prepared as described above
The homodimer was also tested alone or in combination with IGF-I on partial thickness skin wounds. Histological analysis of day 6 wounds showed that PDGF-2 or
Administration of IGF-I alone had no significant differences from controls in terms of connective tissue morphology or epithelial morphology. However, combining PDGF-2 with IGF-I resulted in a 2.0-fold increase in the width of the new connective tissue layer and a 20% increase in epithelial thickness 6 days after surgery. On day 9, PDGF-2 alone reduced both new connective tissue and epithelial thickness by 22%.
resulted in an increase in combined with IGF-I
PDGF-2 is present in both neoconnective tissue and epithelial layers.
resulted in an increase of 75%. PDGF-2/IGF-I
The treated wound had distinct areas of light polarization, indicating that the wound had more mature connective tissue compared to wounds treated with PDGF-2 alone or no treatment.

Therefore, recombination using the above animal models
Application of PDGF-2 to the wound gave similar results when purified human PDGF was combined with recombinant IGF-I. The combination of recombinant PDGF-2 and IGF-I resulted in a dramatic increase in the number of new fibroblasts and the rate of collagen synthesis, in untreated animals or with recombinant PDGF-2 or
It was associated with increased dermal and epidermal hyperplasia (2.5-fold total increase) compared to IGF-I treatment alone.

These results indicate that both recombinant PDGF-2 homodimer and naturally purified human PDGF
This suggests that they interact synergistically when administered locally to a wound.

Dosage To determine the appropriate dose of purified PDGF, we repeated the above experiment, using 2.5 ng per ^mm2 of wound, dispersed in 30 μl of biologically acceptable gel;
Wounds were treated with 5.0 ng, and 10 ng PDGF (calculated as purified PDGF). As a result, the optimal effect is
It was obtained when the PDGF content was 5.0 ng/mm ² or more.

To determine the appropriate dose of purified PDGF + IGF-I, the weight-to-weight ratio of PDGF and IGF-I was adjusted to 1:
Combinations ranging from 10 to 25:1 were evaluated as described above. Optimal results were obtained with ratios of 1:1 and 2:1.

Bone Regeneration To investigate the effect of PDGF/IGF-I preparations on promoting periodontal ligament and/or bone growth, the following experiment was conducted.

First, beagles with naturally occurring periodontal ligament disease were selected using radiographic examination. For cases in which 30% to 80% bone loss was observed, tartar was first removed using an ultrasonic device. A surgical skin flap and root surface smoothing procedure were performed, and the experimental tooth was
Purified PDGF in a pharmaceutically acceptable carrier material, e.g. a commercially available inert gel, e.g. methylcellulose.
and treated with a composition containing IGF-I. 4
One-third of the teeth received control gel alone or purified PDGF alone or IGF-I alone. Tooth and bone block biopsy specimens were taken 2 weeks after surgery and prepared for histological examination using standard demineralization and processing methods.

Results The results of histological analysis of periodontal ligament and bone specimens are as follows:
There were distinct areas of new bone formation adjacent to the root surface of the experimental specimens (i.e., those treated with the PDGF/IGF-I combination), and on the root surface adjacent to this new bone. Deposits resembling cementum were present. New bone was also present on the periosteal surface of the specimen, and an area of dental ankylosis developed in the apical extension of the ligament. A thick layer of osteoblast-like cells and connective tissue lined the new bone with collagen fibers entering the newly formed cementum.

In control specimens, there was no evidence of new bone formation, no new cementum-like deposits were present, and connective tissue was aligned perpendicular to the bone surface to form a cap over the original bone. Looked. Therefore, the results indicated that the PDGF/IGF-I compositions of the present invention enhance bone formation and connective tissue responses.

Other aspects are within the scope of the claims.